CA1312277C - Avidin- and biotin-immobilized reagents, analytical elements and methods of use - Google Patents

Abstract

AVIDIN- AND BIOTIN-IMMOBILIZED REAGENTS, ANALYTICAL ELEMENTS AND METHODS OF USE
Abstract of the Disclosure Reagents have been prepared from water-insoluble polymeric particles to which are covalently attached avidin, biotin or an avidin or biotin deriv-ative. The polymeric particles comprise a polymer on at least the outer surface which is derived from at least one ethylenically unsaturated monomer having a reactive activated 2-substituted ethylsulfonyl, vinylsulfonyl or active halogen atom. Covalent attachment of avidin, biotin or an avidin or biotin derivative is effected either directly or indirectly through these reactive groups. The resulting reagent is useful in analytical elements and various analy-tical methods including agglutination and sandwich assays. The immobilized avidin, biotin or derivative can be used to complex with the corresponding biotin or avidin molecule which may be conjugated to a com-pound of biological interest.

Description

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AVIDIN- AND BIOTIN-IMMOBILIZED REAGENTS, ANALYTICAL ELEMENTS AND METHO~S OF USE
Field of the Inventlon The present invention rel~te~ to water-insoluble reagents which are useful in various ~naly-tical procedures. It a1SQ relates to an~lytic~l ele-ments ~nd methods using these r~Rgent~.
B~ck~round of the Invention There is ~ continuous need in medic~l prOEc-tice, re~esrch and diagnostic procedures for rapid,&ccurate, quRntitative determinations of biological subst~nces which ~re pre3ent in biological fluids at low concentration~. For exsmple, the presence of dru~, narcotics, hormones, steroids, polypeptides, prost~landins or infectious organisms in blood, urine, ~aliva, va~inAl secretions, seminal fluids and other biological fluids has to be determined in an accurste and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, vArious methods have been devised for isolating and ldentify-ing biologic~l substanres employing specific binding reactions between the sub~t~nce to be detected and receptors reactive with thRt sub~t~nce. Radioactive or enzyme label~ have been used to detect the re~ult-ing reactive complex.
One particul~r type of test which h~s been developed is an ~gglutinRtion test which is u~eful for the detection of antigens which have multiple sites for ~ntibody reactivity. In ~uch a test, anti-body molecules can be bound in a suitable fRshion to water-insoluble partlcles. Antibo~y-antigen reaction at multlple sites causes the p~rticles to agglutinate Rnd precipltate. Suitable ~epar~tlon and detection 3s me~ns h~ve been devlsed to make the agglutinste re~d-ily observable, including for example, the use of ``-` 13~2~7 particles containing a tracer material as described in U.S. Patent No. 4,997,772 (issued March 5, 1991) by Sutton, Littlehale and Daniel~on, and in references cited therein.
Another use~ul method for detecting bio-logical substances in fluids is what is known in the art as a "sandwich" assay. Such an assay involves "sandwiching" the compound of interest (such as an anti~en) with two or more receptor molecules (such as antibodies) which complex with the compound at dif-ferent and noninterfering sites. Examples of such assays are described, for example, in U.S. Patent 4,486,530 (issued December 4, 1984 to David et al).
In most sandwich assays, one or more of the receptor molecules are suitably immobilized on an insoluble carrier such as small particles, membranes, plates, test wells or similar objects.
Attachment of antibodies or receptor molecules to insoluble carrier materials has been achieved in the past in a number of ways. Early work relied on adsorption of the molecules, but it was realized that adsorption is generally not a strong method of attachment. Later researchers found that the molecules could be covalently attached by reac-tion of certain functional groups of the molecules with specially designed reactive groups on the car-rier material. For example, proteins have been attached by reacting carboxy groups of particles or supports with an activating compound which renders the groups reactive with amino groups of a protein.
Carbodiimides are examples of useful activating com-pounds.
Avidin is a protein found in egg whites.
Biotin (hexahydro-2~oxo~ thieno[3,43imidazole-4-pentanoic acid), also known as Vitamin H, is a rela-tively small water-soluble molecule. These materials ~a .

~2~

~re known to resct ~peciflcally with each other to form a very strong ~nd st~ble complex in whlch each of the four subunitq of &vldin binds a biotin mole-cule. This strong binding i~ maintained even when either biotin or avidin or both are bound cov~lently to other mater1al~. The reaction hQs been u~ed to enhance ~gglutin~tion of erythrocytes and provided researchers with a means for v~rious biochemical and diagnostic ~tudies.
U.S. Patent 4,298,685 (is~ued November 3, 1981 to P~rikh et al) describes a competitive immuno-a~5~y in which antibodies con~ugated to biotin are allowed to compete with the unknown analyte and ~
known quantity of enzyme-l~beled analyte. The umount of Antibody-analyte complexes is readily determined by insolubilizin~ the complexes by adding avidin sttached to ~ carrier materisl. Avidin is bourld to a solid ~upport such as particles, filter p~per, glass or plastic ob~ect by co~alent attachment, for example covalent attachment to benzoquinone-activated sepharose.
U.S. Patent 4,582,810 (issued April 15, 1986 to Rosenstein) and PCT Publication 84l03358 (pub-lished Augu~t 30, 1984) describe the attachment of avidin to latex particles having free carboxyl ~roups on their surfaces. A~ described therein, the con ventional procedure for covalently attaching avidin to the particles ~nvolve3 the us2 of a water-soluble carbodiimide in sn activation ~tep. While producing reagents, this procedure tends to activate the ex-posed reactive groups of the protein avidin as well as the carboxyl group~ on the particles. The result is intramolecular and intermolecular cro~slinXing or polymerization of avidin, and a si~nificant portion of the reagent is impaired $rom complexation with 1312~ 1 J

biotin. In addition, there may be premature agglu-tination of the insolubilized reagent due to the cross-reactivity of the activating compound. These problems present a serious economic loss as well as an impairment of diagnostic se~sitivity. It has also been evident that carbodiimides provide a reactive intermediate for avidin attachment which is unstable and must be used immediately.
Various o~her reagents have been prepared with particles having reactive groups such as epox-ides, aldehydes, amino groups and diazonium salts.
All of these groups have disadvantages. Epoxide groups are not stable, so that the particles cannot be stored for very long. Particles having aldehyde groups generally tend to agglutinate prematurely.
The aldehyde groups also prematurely oxidize, thereby losing binding activity. Particles with amine groups are like the carboxylated materials by requiring an additional activation step for attachment. Diazonium compounds are unstable and therefore undesirable to work with.
Immunological compounds are immobilized on polymer particles having reactive activated 2-substituted ethylsul~onyl and vinylsulfonyl groups to form useful immunological reagents as described in Canadian Application Serial No. 548,335 filed October 1, 1987 by Sutton and Danielson. In some instances, however, it is not advisable to directly attach immunological compounds to the particles. For example, direct attachment may deactivate the immuno-logical compounds. Yet, it is desired to strongly attach those compounds to the particles.
Hence, reagents which are composed of avidin or biotin covalently attached to a water-insoluble particle would be very useful in diagnostic methods.
However, it would be desirable ~o have such reagents .

~3~ 22~'~
~ 5--which ~re read11y prep~red in an efficient manner and under condition~ which sre not limiting and which do not reduce ~ensitivity or gener~te other undesirable results. It would be particul~rly desir~ble to avoid the use of conventional carbodiimide chemi~try for ett~chment whereby premature crosslinking and agglu-tination ~re prominent. It would also be desirable to have reagent~ for insolubilizing immunological species without directly attaching the species to the insoluble c~rrier mAteri~l. Yet the resulting attachment should be Qtronger th~n that ~chieved throu~h mere adsorption.
Summary of the Invention The problems noted above with conventional rea8ent9 have been overcome with a water-insoluble reagent comprising:
a polymeric particle comprising a polymer on at least its outer surface which is derived from one or more ethylenically unsaturated polymerizable mono-mers, at least one of which monomers hss reactivegroup~ which are selected from the group cons~sting of ~ctivated 2-substituted ethylsulfonyl, vinylsul-fonyl or an ~ctive halogen atom, the p~rticle beln~ covalently ~ttached through the reactive groups on the outer surface of the particle to R specific binding ligand selected from the ~roup consisting of svidin, biotin or ~n avidin or biotin derivative.
This invention also provides an element comprising ~n ~bsorbent c~rrier material having one or more zones, and containin~ in one or more of the zones the water-insoluble reagent described above.
Further, a method for the determination of a compound of biological interest in an aqueous liquid comprises:

~ 3 ~ 7 A. contacting the llquid with the w~ter-insoluble reagent described ~bove, B. formin~ a reaction product of the re~gent with the biological compoun~, ~nd C~ determinlng the amount of the bl~logical compound a~ a result of the presence of the reaction product.
The present invention provide~ stable rea-8ents which c~n be used in ~ v~riety of an~lytical ~nd di~gnostic procedures to gre~t adv~ntage. The polymer partlcles used to prepare the re~gents h~ve re~dily av~ilable function~l groups which readily react with svidin, biotin or ~vidin or biotin deriva-tives. The useful functional groups include an ~c-tivated 2-substituted ethylsulfonyl group, vinylsul-fonyl ~roup or R group cont~ining an active halogen atom. The reaction between ~vidln or an avidin derivative with particles containing these groups can be accDmplished without the need for activQtors such 20 RS c~rbodiimldes ~nd additional activ~tion steps.
Therefore, the problems as~oci~ted with the actlva-tion of carboxy groups (that i5 premature croqslinX-lng and agglutination) are gener~lly uvoided~
In addition, preferred reagents of this invention wherein the particles have reactive vinyl-sulfonyl and activated 2-substituted ethylsulfonyl group~ exhibit further advsnt~ges in that they can be reacted under mild pH conditions and low tempera-tures. Hence, the conditions of att~chment are not - 30 ~s critical ~nd lower temperatures, shorter reaction times and flexible mixing conditions can be employed without sacrificing ~ensitivity. Yet the re~gent of this invention can be used to strongly R~t~ch immuno-logic~l or other specific binding compounds to an insoluble carrier mAterial.

~3~2~77 Detailed Description of the Invention The present invention provides a reagent for use in Qnalytic~l method~ ~nd element~ whereby a detectable complex is obtained u~ing the hi~h speci-fic bindin~ ~ffinity of svidin for biotin. Themethods can quickly provide ~ dQtermin~tlon so that the assay can be performed in a doctor'~ office or in a consumer's home to provide immediate result~. The test can be used to detect the presence or ~bsence of lQ a compound of biologic~l interest in ~n aqueou~
liquid, such ~Q ~ biologic~l fluid.
A compound of biologic~l interest is defined herein aq any biologicAl or chemical compound which h~s one or more ~ites for complexing with a corre~-ponding specific bindin8 receptor molecule. In oneembodiment, the compound of biological interest may be avidin or biotin which can then be detected with the reQgent of this invention containing the corres-ponding receptor therefor. For example, to detect biotin in ~ fluidl ~ re~gent having avidin molecules Qttsched thereto i5 used.
Compounds of biological interest can also be defined as li~ands which specifically complex with R
correspondin~ receptor molecule which iq not biotin or avidin. For ex~mple, the compound could be en immunological ~pecies which is (1) eny sub~t~nce which, when presented to ~n immunocompetent hoqt, will result in the production of e specific ~ntibody cQpable of binding with that subst~nce, or (2) the antibody so produced, which compound perticipates in an anti~en-~ntibody re~ction. In such em~odiment~, svidin, biotin or ~n avidin or biotin derivative i~
~uitably attached to the receptor molecule which reacts ~pecific~lly with the biological compound.

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Representative ligands detPctable with the present invention include prim~ry amines, amino acid3, peptides, polypeptides, proteins, l$popro-teins, ~lycoproteins, drugs, h~ptens, enzymes, steroids, lipids, nucleic acid~, hormones, vitamins, polys~ccharide~, glycolipids, alk~loids, org~ni~ms ~bacteria, protozoa, fun~i, viruses, rickett~ia ~nd the like) and components thereof, blood components, ti~sue and organ antigens and other materials known to one 3kllled in the art. In some instances, the lig~nd i~ ~n ~ntibody which i~ directed again~t a drug, hormone, ~ntibiotic or other compound havlng antigenic properties. Alternatively, the ligand can be an antigenic materi~l (including mono- or multi-v~lent or multideterminant antigens). In stillanother embodiment, the immunological species i9 an antibody which is directed agaln~t another antibody (that i~, an ~nti-antibody). Both monoclonal and polyclonal ~ntibodies can be used, and they can be whole molecules or variou~ fra8ments thereof.
The rea8ent of the present invention is prepared ~y covalently attaching avidin, biotin or R
derivative of either to water-insoluble pulymeric particles of specific composition. The attachment is achieved throu~h amino or sulfhydryl groups of the avidin, or avidin or biot~n derivative which are available for reaction directly with reactive groups on the outer surface of the particles. "Direct"
attachment means that the aYidin or avidin or biotin derivative molecule is directly reacted with the particle groups. Alternatively, the materl~l can be chemically modified to provide reactive sltes for attachment as long as such modi~ication does not adversely affec~ the sites where ~vidln and biotin will complex with each other. For example, biotin ~ 3 1 2 2 7 rl g c~nnot be att~ched directly to such particles, but suitable biotin deriv~tive~ having suitable re~ctive group~ (~uch as succinimidooxycarbonyl, m~leimido-oxycarbonyl or N'-bromo~cetylhydr~zinocarbonyl) can be ~tt~ched to p~rticles pretre~ted with a prot~in such QQ casein to provide ~mine groups thAt react with the biotin deriv~tive. Biotin deriv~ives hsving reactive amine or sulfhydryl group~ can be directly attached to the particles. In ~ddition, avidin, biotin, or ~ deriv~tive of either c~n be "indirectly" attached throu~h a linking moiety which can be a protein, peptide/ polypeptids, diamine or dimercapt~n.
Avidin and biotin derivatives which can be used to prepare the re~gents of this invention in--clude streptavidin, succinylated avidin, monomeric avidin, biocytin ~that is, biotin--N-lysine), biocytin hydrazlde, smine or sulfhydryl derivatives of 2-iminobiotin and biotinyl-~-~minocaproic acid hydr~zide.
Biotin derivatives, such as biotin-N-hydroxysuccinimide ester, biotinyl--aminocaproic acid-N-hydroxysuccinimlde ester, sulfosuccinimidyl 6-[biotin amido~hexanoate, N-hydroxysuccinimide--iminobiotin, biotinbromoacetylhydrQzide, ~-diazo-benzoyl biocytin and 3-(N-maleimidopropionyl)-biocytin, can also be attached to linking proteins a~ter such proteins have been suitably att~ched to the polymeric particles.
The particles used in preparing the present invention comprise one or more polymers each of which i5 prepared from one or more ethylenically unsatur-ated polymerizable monomers which ~re described below in more det~il. At le~st one of such monomers pro-vides the desired reactive groups on at least the 131 ~ ~ r~

surface of the particles. In some embodiment~, the particles are homogeneous, that is, they are composed of the same polymer throughout. In other embodi-ments, the particles can be composed of two or more polymers, for e~ample as core/shell particles (described for example in U.S. Patent No. 4,9~7,772 noted above, and in U.S. Patent 4,401,765, issued August 30, 1983 to Craig et al), or as graft copolymers as described for example in U.S. Patent 3,700,069 (issued October 24, 1972 to Tregear et al).
The polymer}c particles are generally water-insoluble latex particles having an average particle size greater than about 0.01 micrometers.
Preferably they have an average particle size in the range of from about 0.01 to about 5 micrometers.
As described above, the polymeric particles useful in the practice of this invention comprise at least one polymer derived from at least one a, ~ - ethylenically unsaturated polymerizable monomer having one or more of the reactive groups selected from the group consisting of activated 2-substituted ethylsulfonyl, vinylsul~onyl or an active halogen atom.
Monomers having an active halog~n atom include vinyl chloroacetate, vinyl bromoacetate, haloalkylated vinyl aromatics (for example, chloro-methylstyrene or bromomethylstyrene), haloalkyl acrylic or methacrylic esters (for example, chloro-ethyl methacrylate, 3-chloro 2-hydro~ypropyl meth-acrylate and 3-chloropropyl acry~ate) and others known to one skilled in the art. The haloalkylated vinyl aromatics, for example those having active haloalkyl groups of 1 to 3 carbon atoms, are pre-ferred when the active halogen atom is used as the reactive group. Chloromethylstyrene is most preferred.

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A~ noted above, the monomers having active helogen stoms exhiblt m~ny ~dv~nta~e~ over the mate-rial~ of the art~ However, monomers having activated 2-substituted ethylsulfonyl and vinyl~ulfonyl groups pos~ess ~dditional ~dv~nt~ges in that proteins can be attached to the polymers under milder conditions ~nd require les~ process control durin~ manufacture.
This renders m~nufacture more efficient and less costly. A number of repre~entative monomers h~vin~
the requisite pendant ~roups are known in the ~rt, including those di~closed in U.S. Patent~ 4,161,407 (i~sued July 17, 1979 to Campbell) and 4,S48,870 (issued October 22, 1985 to Ogaw~ et al~.
Preferred activated 2-substituted ethylsul-fonyl and vinylsulfonyl monomer~ can be represented by the formula (I):
R O

CH2= C - L- S - R (I) o wherein R is hydrogen or ~ubstituted or unsubstituted alkyl (generally of 1 to 6 carbon atomq, ~uch a5 methyl, ethyl, isopropyl or hexyl.
Preferably, R is hydrogen or methyl.
R is -CH=CHR or -CH2CH2X wherein X
is a leaving group which is displaced by a nucleo-phile or is elimin~ted in the form of HX by treatment with a base (such as halo, ~cetoxy, alkylsulfonyloxy such as methylnulfonyloxy, arylsulfonyloxy such ~-tolyl~ulfonyloxy, tri~lkyl~mmonio, for exam2ple, trimethylammonio salt or pyridinio ~alt). R is hydro~en, substituted or unsubstituted ~lkyl (generally of 1 to 6 carbon atoms as defined for R), or ~ubstituted or un~ubstituted aryl (generelly of 6 to 12 nucle~r carbon atoms, ~uch a phenyl, naphthyl, xylyl or tolyl). Prefer~bly, R is -CH2CH2X.

~ 3 ~ ~ ~ r~ r~

Thi~ group, which i~ an activsted 2-sub~tituted ethyl group, can be su~stituted with ~ny group which does not impair the displacement of the leaving group X.
L i~ a linking group which can be a sub-stituted or unsubstituted alkylene generally having 1to 20 carbon and hetero atoms in the b~ckbone. This definition of ~lkylene is meant to include alkylene groups interrupted or termin~ted with oxy, thio, -NR - [wherein R is hydrogen, substituted or ungub~tituted alkyl of 1 to 6 carbon ~toms (~uch as methyl, chloromethyl or 2-hydroxyethyl~ or sub-stituted or un~ubstituted aryl of 6 to 10 cRrbon atoms (~uoh aA phenyl, naphthyl or xylyl)], o ester (~COO-), amide (-CONH-), urylene (-NHCNH-), sulfonyl (-S02-), carbon~te, sulfon~mide, azo, phosphono or other similar groups. RepreQentative alkylene group~ include methylene, ethylene, i~o-butylene, hexamethylene, carbonyloxyethyleneoxy-carbonylethylene, methylenebis(iminocarbonyl)-ethylene, carbonyloxydodecylenecarbonyloxyethylene, carbonyliminomethyleneiminocarbonyliminoethylene, carbonyliminomethyleneiminocarbonylethylene and other group3 described or suggested by U.S. Patents 4,161,407 and 4,548,870, noted above.
L c~n al~o be ~ubstituted or un~ubstituted arylene ~enerslly having 6 to 12 nuclear c~rbon atoms. Representative arylene group~ include phenylene, tolylene, naphthylene Rnd others noted ln the patents mentioned above. Also included in this definltion of L are divalent ~roups whlch ~re com-bination3 of one or more of eAch of the ~lkylene and arylene groups defined ~bove (for ex~mple, ~rylene-slkylene, alkylene~rylenealkylene ~nd o~hers readily determined by one o ordinary ~kill in the art), as ~ 3 ~

well as such combin~tions which Rre interrupted or termin~ted by one or more amide or estQr groups (for example, c~rbonyliminosrylenealXylene~. Preferably, L is sub~tituted or un~ubstituted phenylenealkylene [for ex~mple, sub~tituted with one or more alkyl groups (~s deflned for R~, alkoxy groups (generally of l to 6 carbon atoms, for example, methoxy, propoxy or butoxy) or halo groups], carbonyliminoarylene-~lkylene (wherein arylene and alkylene are defined above), or carbonyliminoalkyleneiminocarbonylalkylene ~wherein ~lkylene are defined above).
Representative useful monomer~ include m &
~-(2-chloroethy~sulfonylmethyl)styrene, m & ~-[2 tolylsulfonyloxy)ethyl~ulfonylmethyl]styrene, m &
~-vinylsulfonylmethylstyrene, N-[m & ~-(2-chloro-ethylsulfonylmethyl)phenyl]Rcryl~mide, ~nd N-C2-(2-chloroethyl~ulfonyl)ethylformamidomethylJ~crylamide.
The first monomer ls preferred.
One or more of the monomers described above c~n be polymerized individuPlly or in combination to form homo- or copolymers. Alternatively, And prefer-ably, one or more of them are copolymerized with at least one other ethylenically unsaturated polymerlz-able monomer. Generally such monomers provide var-ious desirable properties such as hydrophobicity,dispersibility or other features. Preferred polymers csn be represented by the formula (II):
( A ~x ( B ~ D ~ (II) wherein -A- represents recurring unit~
derived from one or more hydrophobic ethylenically uns~tur~ted monomers, -B- represents recurring units derived from one or more ethylenically unsaturAted monomers having the requisite reactive groups described ~bove, and ~ 3 ~ 2 ~ ~ 3 -D- represents recurring units derived from one or more ethylenically unsaturRted monomer~ which are different th~n those repre~ented by -A- or -B-.
In formul~ (II), x i3 from O to 8bout 9g.9 mole percent, y i9 from ~bout 0.1 to 100 mole per-cent, ~nd z i~ from O to about 20 mole percent.
Preferably, x i~ from ~bout 45 to ~bout 99.5 mole percentl y i9 from about 0.5 to about 50 mole percent, and z is from O to about 10 mole percent.
Monomers from which the -A- recurrin~ units are derived, both in general and in preferred embodi-ments, ~re hydrophobic snd form homopolymers ~hRt ~re insoluble in water. Preferably, these monomers have aromatic groups. RepresentRtive hy~rophobic monomers include, but ~re not limited to, ~tyrene and styrene derivative~ (for example, 4-vinyltoluene, 2,5-di-methylstyrene, 4-t-butylstyrene, 2-chlorostyrene ~nd others known in the art), scrylic and methacrylic acid ester~ and amides (for example, n-butyl acry-late, propyl methacrylate, methyl acrylate, methylmethscrylate, ethyl meth~crylate, 2-ethylhexyl meth-~cryl~te, N-phenylacrylamide and others known in the art), ~crylonitrile and vinyl acet~te.
This polymer can be crosslinked, if desired, in uny suitsble fashion~ One method i3 to incor-porflte a smsll amount, that is up to about 10 mole percent, and preferably from about 0.3 to ~bout 5 mole percent, of ~ monomer having two or more ethylenic~lly unsaturated polymerizable groups.
The~e monomers are included ~mon~ the hydrophobic monomers from which A is derived. Representative monomerq are described in Research Disclosu2e, publication 19551, July? 1930, p~ge 304, And include for ex~mple, divinylbenzene, ethylene dimethacrylate, N,N'-methylenebisRcrylamide, 2,2-dimethyl-1?3-~l3~2~7rJ~

propylene diacrylate, ~llyl acryl~te, ethylidyne trime~hacryla~e and ethylene diacrylate. Cross-linking with such monomers, however, reduce~ the swellability of polymers, especislly of the core of core/shell polymers caused by the organic solvent used in preferred techniques for imbibing tracer msterials into polymeric particles. Therefore, crosslin~ing is generally limited to small amount~ as required to impart water-insolubility.
Preferred monomer~ from which the -A- recurring unit~ are derived are vinyl aromatic monomers, especially styrene and styrene derivatives.
The monomers from which the -B- recurrin~
units are derived are those hflvlng the reac~ive groups described above.
Monomers from which the -D- recurring units are derived include monomers different than those from which -A- and -B- are derived. Specifically, the -D- recurring units are derived from monomers which impart aqueous dispersion stability to the particles or other properties. Representative monomers include, but are not limited to, anionic monomers such as sodlum 2-acrylamido-2-methylpro-panesulfonate, ~crylic acid, methacrylic acid, 2-carboxyethyl acrylete, styrene sulfonic acid, pota~sium salt and m & p-carboxymethylstyrene and other ethylenic~lly unsaturated polymerizable sulfonates, carboxylates, ~ulfates and phosphonates, other hydrophilic but nonionic monomers, such as 2-hydroxye~hyl ~crylate and 2-hydroxyethyl meth-acrylate and other~ known to one skilled in the ~rt.
Preferred monomers from which the -D- units are derived are acrylic acid, methacrylic acld, ~odium 2-acrylamldo-2-methylpropanesulfon~te, m &
P-carboxymethylstyrene and ~-styrenesulfonic acid, potassium salt.

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Representative polymers of the monomers described above include the following: polY(m &
~-chloromethylstyrene~, poly(styrene-co-m & ~-chloromethylstyrene-co-2-hydroxyethyl acrylate) (67:30:3 molar ratio), poly(styrene-co-m & ~-chloroethylsulfonylmethylstyrene) (95.5:4.5 molar ratio), poly{styrene-co-N-[_ & p~(2-chloroethyl-sulfonylmethyl)phenyl]acrylamide} (99.3:0.7 molar ratio), poly(m & R-chloromethylstyrene-~Q-methacrylic acid)(95:5, 98:2 and 99.8:0.2 molar ratio), poly-(styrene-co-m & P-chloroethylsulfonylmethylstyrene-co-methacrylic acid)(93.5:4.5:2 molar ratio), poly{styrene-co-N-~_ & ~-(2-chloroethylsulfonyl-methyl)phenyl]acrylamide-co-methacrylic acid}(97.3:0.7:2 molar ratio), and poly(styrene-co-_ & p-chloromethylstyrene)(70:30 molar ratio).
As noted above, the particles useful in the practice of this invention can be homogeneously com-posed of one of the polymers described above, or a mixture thereof. Alternatively, the polymers described above can be an outer graft or shell of a grafted copolymer or core-shell particle, respec-tively. Useful core-shell polymers are descrlbed, for example, in U.S. Patent No. 4,997,772, noted above.
The polymeric particles can be prepared using any suitable polymerization technique, includ-ing emulsion (including batch, semi-continuous and continuous) and suspension polymerization techniques, graft copolymerization, and others known to one skilled in the polymer chemistry art. Emulsion polymerization is preferred as it can be used to provide particles without the use of aurfactants or emulsifiers as described for example in U.S. Patent 4,415,700 (noted above) and Research Disclosure publication 15963 (July, 1977). Research Disclosure ..

~ 31~277 is a publication available from ~enneth Mason Publications, Ltd., The Old Harbourmaster's, 8 North Street, Emsworth, Hampshire P010 7DD, England.
Continuous emulsion polymerization is the most pre-ferred technique, as described in the noted R~search _sclosure publication. Other details of preparatory methods can be found in ~.S. Patents 4,161,407 and 4,548,870, noted above.
Staged emulsion polymerization can be used to provide a core-shell polymer composed of two dif-ferent polymers. Emulsion polymerization of the core is carried to substantial completion by continuously adding reactants to a reaction vessel under standard conditions. Monomers and catalysts needed to make the shell polymer are then continuously added to the vessel containing the latex of the core polymer. In this manner, the shell has a definite known composi-tion rather than being a mixture of core and shell monomers. Representative details of preparing the core-shell polymeric particles useful in this inven-tion are provided in U.S. Patent No. 4,997,772, noted above.
The general procedure for preparing the reagent of this invention includes covalently attach-ing avidin, biotin or an avidin or biotin derivative to the particles using generally known reactions.
With the active halogen atom, 2-substituted activated ethylsulfonyl and vinylsulfonyl groups, avidin or an avidin derivative can be directly attached to the particles. Biotin or derivatives thereof can be attached indirectly as noted above. Generally, the polymer particles are mixed with the material to be attached in an aqueous buffered solution (p~ gener-ally from about 7 to about 10~ and a concentration of from about 0.01 to about 40 weight percent polymer particles (preferably from about 0.01 to about 10 A

_lg_ weight percent). The amount of avidin, biotin or a derivative of either is at a wei~ht ratio to polymer of from about O.l:1000 to about 1:10, and preferably from about 1:100 to about 1:10. Mixing is carried out at a temperature in the range of from about 5 to about 50C, and preferably at ~rom about 5 to about 40C, for from about 0.5 to about 48 hours. Any suitable buffer can be used. The details of a repre-sentative preparatory procedure are illustrated in Example 1 below.
The procedural details of direct and in-direct attachment of avidin, biotin or derivatives thereof are known in the art.
The polymeric particles used in this inven-tion can have a detectable tracer material associated therewith i~ desired. A tracer is a material which is detectable with the unaided eye or with appro-priate equipment and techniques. The tracer material can be inside or outside of the particles. In one embodiment where the reagents are used in an agglu-tination assay, the tracer is preferably inside the particles. Useful tracers include, but are not limited to, radioisotopes which emit gamma rays, fluorescent compounds or dyes, bioluminescent com-pounds, chemiluminescent compounds, chromogens such as dyes and dye-formers which absorb in the visible or ultraviolet region of the electromagnetic spec-trum, and others known to one skilled in the art.
Particularly useful reagents having detectable tracers include those having core/shell polymers in which the tracer is in the core only. U.S. Patent No. 4,997,772, noted above, describes similar reagents. In other embodiments, the tracer i9 on the outside of the particles, either attached to the particles in some manner or as part o~ the avidin or biotin molecules.

~3~2~
-lg-The re~ent of the preQent invention c~n be used in the determination (qu~litative or quantit~-t~ve measurement) of a biological compound in aqueouQ
liquidq. This determination can be made by merely determining the presence or absence of the compound, or by qusntltstively determining the amount of com-pound. In p~rticular, the invention can be used to asQay biological fluids of ~nimals, humans or plants but preferably of hum~ns. Such flulds include, but are not limited to, whole blood, plasma, sera, lymph, bile, urine, spin~l fluid, seminal fluid, lscrimal fluid, v~glnal secretions, sputum, perspiration and the like as well as stool specimens. I~ is also pos-sible to assay fluid preparations of human or animal tissue such as skeletal muscle, he~rt, kidney, lungs, brsins, bone marrow, ~kin and the like.
The present invention can be used to deter-mine avidin, biotin or a derivative of elther which ls reactlve wlth the corresponding moiety which is a part of the reagent of this inventlon. For example, the reagent can be comprised of avldln attached to ~
p~rticle, ~nd the compound to be determined is bio-tin or a deriv~tive thereof with which the avidin is reActive .
Alternatively, the biological compound is a llgand other than avidln or biotin, such as an anti-body or antigen, which has one or more sites for com-plexation with one or more receptor molecules. Such receptor molecules ere usually immunologically reec-tive species. At le~st one of the receptor molecules is con~ugated with avidin or biotin. The ligand can be complexed with the receptors, and the entire complex can be insolubilized by reaction of the con~ug~ted avldin or biotin with the reagent of this lnvention. For exemple, the lig~nd can be Strepto-coccus A antigen, an antigen from chlamydial or 131~

~onococcal organisms, HTLV antigens or antibod~es (for example, HTLV-I or HTLV-II), HIV antigens or ~ntibodie~ (for example, HIV-I or HIV-II), thyroid stimulst1ng hormone, ~polipoprotelns, human chorionic gonadotr~pin, leutinizing hormone, c~rcinoembryonic anti~enl hepatiti3 ~ntigen, herpe~ viruses Qnd other biological ~nd chemical compounds.
The rea~ent can be used in competitive bind-in~ immunoassays. Either bound (th~t is, complexed) or unbound (that is, uncomplexed) labeled materials can be determined. Physical separation of bound and unbound materials, if desired, can be carried out using any suitable sep~r~tion technique~ In using the an~lytical element~ de~cribed below, either verticfll or horizontal separ~tion can be u~ed.
In another embodiment, the rea~ent c~n be used in what are known in the art a5 immunometric ass~ys, for example, "~andwich" asssys. The details of such assays are provided in U.S. Patent 4,486,530 ~noted above). The reagent of the present invention is useful in such R~says where the ligand to be determined has two or more epitopic sites Çor im-munological reaction with two or more receptor mole-cules. The receptor molecules can be the same or different. The receptors ~re capable of immuno-logically reacting with the ligand ~t different sites. The result of the method is the formation of a ternAry complex of the two distinct receptors with the ligand. At least one of the receptors is co-valently ~ttached to biotin or avidin (preferablybiotin). The corresponding avidin or biotin molecule is att~ched to the particles described herein. The ternary complex is insolubilized when the avidin and biotin react, ~nd the resulting insolubilized complex 3s can be ~eparated from unreacted material. The p~r-ticles can be suitably labeled for detection, or one ~ ~ ~ 22~

or more of the receptor molecules can be sultAbly labeled, such AS with an enzyme. In a preferred immunometric asssy, both receptors ~re distinct antibodies directed ~gainst an antigen, one of which ~ntibodies is enzyme-labeled. They can b~ the 3ame or different antibodies, whole or fragments, mono-clonsl or polyclonal.
The method of this invention can be used in either solution or dry asssys. By ~olution ~ssay is meant that the reagents are used in liquid suspen-sion In dry assays, the reagent is incorporated in a dry ~nslytical element. The ~implest element can be composed of ~n absorbent carrier material, for example, a thin sheet of 8 self-~upporting absorbent or bibulous material, such ~s filter paper or strips, which has one or more zones, at least one zone con-tAining the reagent of this invention. Other zone~
can be used to contain other useful reagents. Such elements are known in the art ~s test strips, di-agnostic elements, dip sticks or diagnostic ~gents.
Useful absorbent carrier materials areinsoluble and maintain their structur~l integrity when exposed to w~tPr or biological fluids such as whole blood or serum. Useful elements cRn be pre-pared from paper, porous particulate structures,porous polymeric films, cellulose, glass fibers, woven and nonwoven fabrics (synthetic and nonsyn-thetic) and the like. Useful materials and proce-dures for makin~ such elements are well known in the art.
Preferably, the absorbent carrier materi&l of the dry analytical element of this invention is a porous spreading zone. Thi3 zone can be self-supporting (that is, composed of ~ materi~l rigid enough to maintain its integrity), but prefer~bly it ~122~7 is c~rried on ~ separate ~upport. Such a ~upport can be any suitable dimensionally stable, and preferably, nonporous and transparent (th~t is, radiation trans-mis~ive) materi~l which transmits electroma~netic radi~tion of a wavelength between about 200 and about 9~0 nm. A support of choice for a particular element should be compatible with the intended mode of detec-tion (fluorescence, transmission or reflectance spec troscopy). Useful supports can be prepared from paper, metal foils/ polystyrene, polyesters, poly-carbonateg or cellulose esters.
The porous 3presding zone can be prepsred from any suit~ble fibrous or non-fibrous material or mixtures of elther or both. The void volume and aver~ge pore size of this zone can be varied depend-in8 upon the use intended. Useful spreading zones can be prepared using materials and procedures described, for example, in U. S. Pstents 4,292,272 (issued September 29, 1981 to Kita~ima et ~1), 3,992,158 (issued November 16, 1976 to Przybylowicz et al), 4,258,001 (issued M~rch 24, 1981 to Pierce et al~ and 4,430,436 (issued February 7, 1984 to KoyamA
et al) ~nd Japanese Patent Publication 57(1982)-101760.
The elements can have two or more discrete zones, eith~r in the same lsyer or superimposed. At least one of the zones ig preferably a porous spread-ing zone. The other zones can be reagent zones or registration zones as those zones are known in the art, additional spreading zones, r~diation-blocking or filter zones, subbing zones or barrier zones. The zones are gener~lly in fluid contact with each other, meaning that fluids, soluble reactive msterisls and soluble re~ction products can pa5s or be transported between superposed regions of ad~acent zones. In other word3, when the element is contacted wi~h fluid, the reagents within the element become mixed and c~n readily interact Preferably, each zone i~
separately coated layer, although two or more zones can be aeparate aress in a ~ingle la~er of the element.
Preferably, the rea8ent of thi~ invention i~
used to dete~t 8 multivalent ligand such as the hormone, human chorionio gonadotropin (hCG), the pre3ence of which in a woman's urine can be an early indicator of pregnsncy. An assay for hCG is demon-strated in Example 1 below. This embodiment relating to hCG i5 presented for illustrative purposes, but it will be understood that the scope of this invention is not 90 limited. A biological sample (usually urine) suspected of containing the hormone is col-lected from a patient and contacted with the reagent of this invention. If the li~and is present, a reac-tion product is formed between the ligand and one or more receptor molecules therefor. One o$ the recep-tor molecules can be conJugated to either avidin or biotin which reacts with the rea8ent of this inven-tion. For example, the reagent can have avidin molecules attached to the particles, which molecules react with biotin molecules which are con~ugated with a receptor molecule (such as an antibody) for hCG.
The amount of ligand is thereby determined by measur-ing the presence or amount of the resulting reRction product which ~s immobilized with the particles.
Generally such detection is carried out by measuring the amount of detectable tracer in the complexed or uncomplexed materisls.
The method of this invention can also be c~rried out in A di~pos~ble test device wherein a filtration membrane is used to separate complexed .

13122 ~

materials from uncomplexed materialæ. Such a device can have one or more wells into which a test sample containing a ligand is added for reaction with the appropriate reagents including the reagent of this invention. An example of such a test device is described and claimed in copending and commonly assigned U.S. Patent No. 4,833,087 (issued May 23, 1989). Other variations of useful test devices would be within the purview of an ordinary worker skilled in the art.
The following examples illustrate the practice of the present invention.
Example 1: Preparation of a Reagent This example illustrates the preparation of a polymer latex and the attachment of avidin in the preparation of a reagent of the present invention.
PreparatiQ~__f Polymer Latex The three solutions outlined below were continuously added to a 1365 ml vessel containing deoxygenated water at 80C at the indicated rates:
Solution 1: Styrene (739 g), m & ~-(2-chloroethylsulfonylmethyl)styrene (82 g) and l-dodecanethiol (8.2 g) at 2.5 g/min. for 380 minutes.
Solution 2: Ammonium persulfate (19.7 g) and distilled, deoxygenated water (1152 g) at 2.14 g/min.
for 380 minutes.
Solution 3: Sodium pyrosulfite (9.9 g~ and distilled water (1152 g) at 2.27 g/min. for 380 minutes.
After 380 minutes, the reaction was stopped, yielding about 1218 g of latex at 33.4% solids. The latex was dialyzed for 3 days to yield a latex having 27.3% solids and a pH of 5. This latex was diluted to 13.5% solids. NMR analysis confirmed a 96:4 molar ratio of styrene to the second monomer. The result-ing latex particles had an average diameter o~ about ~ ~ 2~'77 0.67 ~m as measured by transmission electron microscopy.
Covalent Attachment of Avidin A sample (0.75 ml~ of the latex described above was diluted to 20 ml with borate bu~fer (50 mmolar, pH 8.5~ and avidin (5 mg, Sigma Chemical Co.) was subsequently added. The resulting suspension was agitated in an end-over-end fashion at 37C for 18 hours, followed by centrifugation. The supernatant was discarded and the particles washed once with buffer by centrifugation and resuspended in lO ml borate buffer. Biotin binding analysis (that is, titration with tritium labeled biotin) indicated that avidin had been covalently attached to the particles (7 x 10 6 molar binding sites per 0.3% bead suspen-sion) to form a reagent of the present invention.
Example 2: Preparation of a Larger ~ized Rçagent.
A reagent was prepared similar to that pre-pared in Example 1 except the polymeric particles in the latex had an average diameter of about 2.5 ~m.
A sample (0.65 ml at 15.5% solids) of purified latex was diluted to 10 ml with borate buf~er (50 mmolar, p~ 8.5) and avidin (l mg, Sigma Chemical Co.) was added. The resulting suspension was agitated end-over-end at room temperature for 24 hours.
Radiolabeled tracer analysis (avidin labeled with 125I~ indicated 84% of avidin was covalently bound to the particles following washing of the beads three times.
~xample 3: Use of Reagent in the Determination of Human Chorionic Gonadotropin This example demonstrates the use of the reagent of this invention in an asæay to determine hCG. This example is taken from U.S. Patent No.
4,870,007 (issued September 26, 1989) 13~ 227 ~

date herewith by Smlth-Lewis and entitled "Use of Immobilized Biotinylated Receptor in Te~t Device, Kit ~nd Method for Determining a Ligand".
A test device as described in the ~uat mentioned patent ~pplication was used to determine hCG in R urine sRmple in the following m~nner. This device contsined a filter membrane consisting of a commercially available nylon membrAne co~ted with succinylated cssein. A con~ugate (3 ~g) of biotin and a monoclonal antibody directed to hCG was incor-porated into the sample well of the test device in a costin~ with poly(acrylamide) (60 ~g). A buffer, ~-~N-morpholino)propanesulfonic ~cid (2 mg, pH 7.2), was incorporated in a different location in ~he sample well.
A urine ~ample, prefiltered to remove impurities, and containing About 50 mI.U./ml of hCG, was added to the sample well followed by ~ddition of a second monoclonal antibody to hCG which i5 labeled with horseradish peroxidAse (40 ~1 of a 10 molar ~olution). A complex of sntigen (hCG) and the two Antibodies w~s formed in solution during A one minute incub~tion period at room temperature.
A sample of the immunoreactive re~gent o$
this invention was then added to the well cont~ining the complex. This sample contained 40 ~1 of a 0.42% dispersion of poly[styrene-co-m & P-(2-chlvro-ethylsulfonylmethyl)styrene] pArticles to which avidin was attached. After this addition, fluid in the well was allowed to drain through the membrRne of the test device~ snd a wash solution cont~lning 0.1 molar sodium phosphate (200 ~1) and sodium dodecyl-sulfate (10 mmolar) was added. A dye-providing com-position (40 ~1) was then added. It was prepared Rccording to copending and commonly ~signsd 1~2~7 -~7-Canadian Patent Application Serial No. 569,195 entitled "Dye-providing Composition, Diagnostic Test Kit and Their Use in Method for Ligand Determination Using Peroxidase Labeled-Receptor". This composition comprised 2-(4-hydroxy-3,5-dimethoxyphenyl)-4,5-bis(4-methoxyphenyl)imidazole le~co dye (0.005% in 40 ~1 solution), poly~vinyl pyrrolidone~ (1%), diethylene-triaminepentaacetic acid (10 ~molar) chelating agent, 4'-hydroxyacetanilide (5 mmolar) electron transfer a~ent, hydrogen peroxide (10 mmolar) in an aqueous solution buffered to pH 7 with æodium pho3-phate with a minor amount of methanol. After two minutes of reaction, a detectable dye was formed in the complex retained in the sample well on the membrane. The amount of dye was measured by convert-ing the measured reflectance to transmittance (DT) using conventional equipment and the Williams-Clapper transform (J. Opt. Soc. Am., 43, p.595, 1953). The amount of measured dye was an indication of the pre-sence of hCG in the urine sample tested.
Example 4: Preparation of Reagent Using Particles Having Reactive Chloromethyl ~roups Poly(styrene-co-m & ~-chloromethylstyrene, (77.3:22.7 molar ratio) particles having an average particle diameter siæe of 2.9 ym were prepared using a continuous emulsion polymerization procedure similar to that described in Example 1 above. A
sample of the resulting latex (0.82 ml, 12.2% solids~
was diluted with 20 ml of 0.05 molar borate buffer (pH 8.5, containing 0.01% thiomersal germicide).
Avidin (5 mg> was added to the diluted latex and the resulting suspension was rotated end-over-end at 37C
for 24 hours. Thc avidin was thereby attached to the particles through the chloromethyl groups on the ..~

~3~22~7 psrticle surfaces. Biotin binding capacity, as measured by the procedure described in Example 1, was 1.5 x 10 molar sites per 0.3% of diluted particle ~u~pension.
Example 5: Preparation of Reagent H~vlng Biotin Attached to the P~rticles ~nd Use ln an AssaY for hCG
This example demonstrates the preparatlon of an immunoreactive rea~ent of thls invention whereby biotin i9 attached to water-inqoluble polymeric par-ticleY, ~nd the use of the reagent in an ~s~ay for hCG.
PreParation of Rea~ent A solution of borate buffer (50 ml, 0~05 molar, pH 8.5) contsining 0.01% thiomersal germicide was placed in a centrifu~e tube, followed by the addition of a ~olution (6 ml) o$ casein ~1 mg/ml) in deionized dis~illed water. The tube wa~ capped and ~haken vigorou~ly, then an aqueous dispersion (1.23 ml of 12.22% solids~ of poly(styrene-co-m & ~chloro-methyl~tyrene)(77.3:22.7 molar ratio~ polymeric particles (average size about 2.7 ~m) wa3 added, snd the tube was capped and rot~ted end-over-end for 24 hours at 37C. The resulting particles had casein attached to the outer surfaces through the resctive chloromethyl ~roups.
The rasein-particle rea~ent waq then washed with glycine buffer ~0.1 molar, pH 8.5) containing 0.01% thiomersal, and then resuspended in glycine buffer (0.1 molar) containin~ 0.01% thiomersal to produce a disper~ion (0.3~ solids~ of a casein-psrticle reagent.
A sample of the dispersion ~u~t described (100 ml) was centrifuged, washed with sodium bicsrbonate solution (80 ml, 100 mmolar), centrifuged ~ 3 ~ 2 2 ~ ~

again at about 15,000 rpm for 5 to 6 minutes, and resuspended in sodium bicarbonate (100 mmolar) to produce a 100 ml bead suspension. This suspension was treated with a mixture of biotin esteri~ied with N-hydro~ysuccinimide (10 mg), and allowed to incubate for about 90 minutes. The resulting product was centrifuged, washed with sodium phosphate buffer (200 ml, 50 mmolar, pH 7.2) containing diethylenetriamine-pentaacetic acid (5 ~m), centrifuged again and resuspended in sodium phosphate (100 ml, 50 ~molar, pH 7.2, 0.3~/O solids).
Assav for hCG
A three-well test device like that described in U.S. Patent No. 4,870,007 of Smith-Lewis, noted above, was pretreated by contacting the nylon microporous filter in two of the wells with a solution of succinylated casein (2% solids). The membrane in the third test well was not 90 treated.
A stock solution of hCG (GC-10 chorionic gonadotropin from Sigma Chemical Co.) was prepared with phosphate buffered saline solution (50 mmolar sodium phosphate, 150 mmolar sodium chloride, pH 7) to comprise 5000 mI.U. hCG/ml for use as a test solution.
A sample (150 ~l) of this test solution was placed in each of the two casein-treated test wells of the test device, and the following reagents were immediately placed in each of the three wells:
a) 40 ~1 of the biotinylated reagent described above, diluted l:10.
b) 40 ~1 of an avidin-anti-hCG conjugate diluted 1:200. This conjugate was prepared from a purified ~onoclonal antibody against ~-hCG
(Immunoresearch) coupled to thiolated avidin by the thiol/maleimide procedure of Chen et al, Clin. ~hem., ~3~22~'~

30(9), pp. 1446-1451 (1~84) having 0.01% thiomersal added. The solution was characterized a~ hRving an optical density of 0.072 at 280 nm before dilution.
c) 40 ~1 of a peroxidaQe labeled mono-clonal antibody against hCG diluted 1:250~ This con~ugate w~s prepared by coupling the ~ntibody to horseradish peroxidase es~entially as described by Chen et al, suPrs. The solution comprised 0.49 mg/ml of sntibody before dilution.
The mixture in each test well W~Q allowed to incubate for 30 seconds, then fluid was drained through the membrane, and each well was wa~hed with 8 drops of a solutlon of sodium phosphate (100 mmol~r) and sodium dodecyl~ulfate (10 mmolar). To each well 5 W8S then added one drop of a dye composition prepared by mixin~ the followin~ solution~ A and B in a ratio of 500 ml of A to 5 ml of B.
Solution A:
sodium phosphate buffer (lOOmmolar, pH 7), 1% poly(vinyl pyrrolidone) (MW=40,000), diethylenetriaminepentaacetic acid (5 mmolar) chelating agent, 4'-hydroxyacetsnilide (5 mmolar) electron transfer agent, hydrogen peroxide (5 mmolar), ad~usted to pH
of 7 with sodium hydroxide.
Solution B:
Spectra grade methanol saturated with the leuco dye 4,5-biQ(4-methoxyphenyl)-2-(3,5-~imethoxy-4-hydroxyphenyl~imidazole.
After incubation for 15 seconds, the fluid was drained through the membrane in each test well, and the color formed on the membrane wa~ observed after another three minutes. The color formed was indicative of the presence of hCG ~n the test solution.

13 ~ ~ 2 s ~

The assay described above was repeated twice except that the fluid was drained in each test well ~fter 15 minutes incubstion, followed by color obser-vation after one or two minutes. Also, ln the third assay, the biotinylated rea8ent di~persion wa~ used undiluted. The following table shows the results from these asQ~yS. The~e d~ta show that ~ positive color ls generated in the test wells containing the hCG te~t solution. However, in the third well into which no hCG was added, there WRS essentially no color $ormat10n.

3~22~7rl S ~q . ~ ~ ~ CO
I
:~: ~ s a~ x I I l I I I
ao GO ~ C: o ~ ~ 4 o~ . E~
O C I I I ~ C _~ ~ C ~ ~ C C
c~l l l l 5 D~ a ~

~ I I I ~ I o m I I I ~ ~ ~ I I I
C ~ D~
al ~ U C ~ C P' a ~ e a~
.D ~ G~ E e E E3 E3 e ~ E~ o o o u~ C ~0 , e ~; ~
~ G~ a~ a~ o ~ ~ O ~ Q~ O
~ ~ ~ Z ~ ~ Z ~ ~ C
C~ o) ~ ~
P~ ~ o 0 ~ * ~ #
D~ ~ ~ ~ ~ ~ ~ ~ ~ ~
¢

13~ 227 ~

~xample 6: Use of Reagent to Determine Leutinizing Hormone (LH) Immobilization of Avidin on Particles A solution (50 ml, 0.05 molar, p~ 8.5~ of borate buffer containing thiomersal germicide (0.01%) was placed in a polypropylene centrifuge tube, and to it was added 6 ml of a solution of egg white avidin (6 mg, Sigma Chemical Co.) dissolved in 6 ml of deionized distilled water. The tube was then capped and shaken vigorously, followed by addition o~ 1.35 ml of a dispersion (15.5% solids) of polyCstyrene-co - m & p-(2-chloroethylsulfonylmethyl)styrene]
~95.5:4.5 molar ratio) beads (average size of 2.54 ~meters) and rotation end-over-end for 24 hours.
The polymer beads having avidin covalently attached thereto were washed with glycine buffer (0.1 molar, pH ~.5) containing 0.01% thiomersal, and then resuspended in glycine buffer (0.1 molar) containing 0.01% thiomersal to produce a dispersion containing an insoluble separation specific binding reagent (0.3% solids).
Assav for LH
A test device having three test wells like that described in U.S. Patent No. 4,870,007 of Smith-Lewis (noted above), having a 5 ~m mesh nylon filter membrane which had been pretreated with casein in each well, was used in this assay. Also used in the assay were: a biotinylated antibody to LH (0.044 mg/ml in phosphate buffered saline solution) prepared similarly to the biotinylated antibody to hCG
described above, a horseradish peroxidase labeled antibody to LH (Q.0015 mg/ml in phosphate buffered saline solution containing 0.5% bovine serum albu-min), and the reagent described above comprising avidin on beads (concentrated to 0.9% solids, pH 8.5).

~Q

~L 3~ 227 ~

Several urine samples were tested in thi~
assay:
(a) a sample collected the 13 th day of a womanls menstrual cycle containing 18 mI.U. LH/ml, and (b) a sample collected the 14th day of the woman's menstrual cycle containing 64 mI.U. LH/ml.
Both of these samples were taken from the same person and the LH content was determined by an LH radioimmunoassay kit available from Diagnostic Products Corporation.
A mixture of urine sample (a) (200 ml), the peroxidase-labeled antibody (35 ~1) and the bio-tinylated antibody (lO ~l) was prepared and incu-bated at room temperature for two minutes. The insolubilized avidin reagent (40 yl) was then added to the mixture and incubation was continued for another five minutes. The mixture was then trans-ferred to one of the test wells of the test device, and fluid and unreacted materials were drained away leaving insolubilized ternary complex formed during incubation on the filter membrane. The remaining insolubilized product was then washed twice with phosphate buffered saline solution (125 ~1) containing 0.1% TWEEN 20T~surfactant (a sorbitan monolaurate nonionic surfactant available from ICI
Americas, Inc.), filtered again and then contacted with a dye-providing solution (50 ~1, pH 7) like that described in Canadian Patent Applica~ion Serial No. 569,195 of McClune et al (noted above). This solution is prepared with 5 ml of 4,5-bis(4-methoxyphenyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)imidazole leuco dye in methanol (15 mg/ml), mixed with 500 ml of a buffered solution of poly(vinyl pyrrolidone) (1% in 100 mmolar sodium phosphate), diethylenetriaminepentaacetic acid (5 mmolar) chelating agent, hydrogen peroxide ~5 mmolar) and 4'-hydroxyacetanilide (5 mmolar) electron trans-fer agent.
*Trade Mark Urine ~ample (b) was similarly assayed using 8 Yecond test well of the test device.
In both test wells, ~ color wa~ seen on the filter membrane wlthin five minutes. The color in the first well was light pink in color where~s the color in the second well was bright red in color.
Example 7: AsssY for StrePtococcus A Anti~en This e~Rmple ~emonstrates the preparation and use of a resgent of thi3 invention to determine StreptGcoccus A antigen in a fluid s~mple. It also demon~trates that this reagent i~ not useful for the determinQtion of Streptococcus B antigen.
The reagent was prepared by attaching svidin to poly(styrene-co-m & ~-chloromethylatyrene-co-2-hydroxyethyl acrylate) (69:30:1 molar rfltio)particles (0.69 ~meters average size) using ~
procedure 9 imilar to that described in Ex~mple 1.
The pQrticles sre Qlso prep~red using ~n emulsion polymeriz~tion procedure like that described in Example 1.
A sample (0.15% solids) of the reagent described above was mixed with a biotinyl~ted antibody to Streptococcus A entigen (20% antibody w/w) and a fluid sample contsining extracted Streptococcus A antigen, and the resulting mixture was incubated for 1 hour. After that time, it wa5 observed that an agglutination reaction hsd occurred indicating that the sntibody had reacted with the antigen, ~nd the resulting complex w~s insolubilized by the reaction of avidin with biotin.
In contrsst, no agglutination w~s observed in a similar assay when Streptococcus B antigen was substituted for Streptococcus A antigen.

~3~ ~2~

The invention hR~ been described in det~il with p~rticul~r reference to preferred embQdiments thereof, but it will be underqtood th~t variations snd modific~tion3 csn be effected within the spirit and ~cope of the invention.

Claims (30)

1. A water-insoluble reagent comprising:
a polymeric particle comprising a polymer on at least its outer surface which is derived from one or more ethylenically unsaturated polymerizable mono-mers, at least one of which monomers has reactive groups which are selected from the group consisting of activated 2-substituted ethylsulfonyl, vinylsul-fonyl or an active halogen atom, said particle being covalently attached through said reactive groups on the outer surface of said particle to a specific binding ligand selected from the group consisting of avidin, biotin or an avidin or biotin derivative.

2. The reagent of claim 1 having a detectable tracer material associated therewith.

3. The reagent of claim 2 wherein said tracer is contained within said particle

4. The reagent of claim 1 wherein said specific binding ligand is avidin or a derivative thereof.

5. The reagent of claim 1 wherein said specific binding ligand is biotin or a derivative thereof.

6. The reagent of claim 1 wherein said polymer is represented by the formula:

wherein -A- represents recurring units derived from one or more hydrophobic ethylenically unsaturated polymerizable monomers, -B- represents recurring units derived from one or more ethylenically unsaturated polymerizable monomers having a reactive group selected from the group consisting of activated 2-substituted ethyl-sulfonyl, vinylsulfonyl or an active halogen atom, -D- represents recurring units derived from one or more ethylenically unsaturated polymerizable monomers other than those represented by -A- or -B-, x is from 0 to about 99.9 mole percent, y is from about 0.1 to about 100 mole percent, and z is from 0 to about 20 mole percent.

7. The reagent of claim 6 wherein x is from about about 45 to about 99.5 mole percent, y is from about 0.5 to about 55 mole percent, and z is from 0 to about 10 mole percent in the defined polymer.

8. The reagent of claim 6 wherein said reactive group is haloalkyl of 1 to 3 carbon atoms.

9. The reagent of claim 6 wherein -B- represents recurring units derived from one or more ethylenically usaturated monomers represented by the formula:

CH2-?-L-?-R1 wherein R is hydrogen or substituted or unsub-stituted alkyl, R is -CH=CHR2 or -CH2CH2X wherein X is a leaving group which is displaced by a nucleophile or is eliminated in the form of HX by treatment with base, and R2 is hydrogen, substituted or unsub-stituted alkyl, or substituted or unsubstituted aryl, and L is a linking group selected from the group consisting of substituted or unsubstituted alkylene, substituted or unsubstituted arylene, a combination of one or more of each of said alkylene and arylene groups, and said combinations interrupted or termin-ated with one or more amide or ester groups.

10. The reagent of claim 9 wherein said -B- recurring units are derived from the defined monomers wherein R is hydrogen or methyl, R1 is -CH2CH2X and L is substituted or unsubstituted phenylenealkylene, carbonyliminoarylenealkylene, or carbonyliminoalkyleneiminocarbonylalkylene.

11. The reagent of claim 6 wherein said -A- recurring units are derived from one or more of styrene, vinyltoluene, ethylene dimethacrylate, butyl acrylate, divinylbenzene, 2-ethylhexyl methacrylate and methyl methacrylate, said -B- recurring units are derived from one or more of m & p-(chloromethyl)styrene, m &
p-(2-chloroethylsulfonylmethyl)styrene, m & p-[2-(p-tolylsulfonyloxy)ethylsulfonylmethyl]styrene, m &
p-vinylsulfonylmethylstyrene, N-[m- & p-(2-chloro-ethylsulfonylmethyl)phenyl]acrylamide, and N-[2-(2-chloroethylsulfonyl)ethylformamidomethyl]acrylamide, and said -D- recurring units are derived from one or more of sodium 2-acrylamido-2-methylpropane-sulfonate, sodium acrylate, sodium 3-acryloyloxy-propane sulfonate, sodium methacrylate, 2-hydroxy-ethyl acrylate, 2,3-dihydroxypropyl acrylate, N-iso-propylacrylamide, acrylamide and acrylonitrile.

12. The reagent of claim 1 wherein said particle comprises said polymer homogeneously throughout.

13. The reagent of claim 1 wherein said particle is a core/shell particle wherein said shell comprises said polymer having said reactive groups.

14. An element comprising an absorbent car-rier material having one or more zones, and contain-ing in one or more of said zones a water-insoluble reagent comprising:

a polymeric particle comprising a polymer on at least its outer surface which is derived from one or more ethylenically unsaturated polymerizable mono-mers, at least one of which has reactive groups which are selected from the group consisting of activated 2-substituted ethylsulfonyl, vinylsulfonyl or an active halogen atom, said particle being covalently attached through said reactive groups on the outer surface of said particle to a specific binding ligand consisting of avidin, biotin or an avidin or biotin derivative.

15. The element of claim 14 wherein said reagent polymer is represented by the formula:
wherein -A- represents recurring units derived from one or more hydrophobic ethylenically unsaturated polymerizable monomers, -B- represents recurring units derived from one or more ethylenically unsaturated polymerizable monomers having a reactive group selected from the group consisting of activated 2-substituted ethyl-sulfonyl, vinylsulfonyl or an active halogen atom, -D- represents recurring units derived from one or more ethylenically unsaturated monomers other than those represented by -A- or -B-, x is from 0 to about 99.9 mole percent, y is from about 0.1 to about 100 mole percent, and z is from 0 to about 20 mole percent.

16. The element of claim 15 wherein -B- represents recurring units derived from one or more ethylenically unsaturated polymerizable monomers represented by the formula:

CH2=?-L-?-R1 wherein R is hydrogen or substituted or unsub-stituted alkyl, R1 is -CH=CHR2 or -CH2CH2X wherein X is a leaving group which is displaced by a nucleophile or is eliminated in the form of HX by treatment with a base, and R2 is hydrogen, substituted or unsub-stituted alkyl, or substituted or unsubstituted aryl, and L is a linking group selected from the group consisting of substituted or unsubstituted alkylene, substituted or unsubstituted arylene, a combination of one or more of each of said alkylene and arylene groups, and said combinations interrupted or termin-ated with one or more amide or ester groups.

17. A method for the determination of a compound of biological interest in an aqueous liquid comprising:
A. contacting said liquid with a water-insoluble reagent comprising:
a polymeric particle comprising A polymer on at least its outer surface which is derived from one or more ethylenically unsaturated polymerizable mono-mers, at least one of which has reactive groups which are selected from the group consisting of activated 2-substituted ethylsulfonyl, vinylsulfonyl or an active halogen atom, said particle being covalently attached through said reactive groups on the outer surface of said particle to a specific binding ligand consisting of avidin, biotin or an avidin or biotin derivative, B. forming a reaction product of said reagent with a receptor molecule for said specific binding ligand, which receptor molecule is or will be com-plexed with said biological compound, and C. determining said biological compound as a result of the presence of said reaction product.

18. The method of claim 17 wherein said reagent comprises avidin, or a derivative thereof, which is reacted with said receptor molecule which is conjugated to said biological compound.

19. The method of claim 17 wherein said reagent comprises avidin, or a derivative thereof, which is reacted with said receptor molecule which is conjugated with an immunoreactive species capable of immunological reaction with said biological compound.

20. The method of claim 19 for the determination of hCG in said liquid wherein said immunoreactive species is a first antibody against hCG, find wherein said method is carried out with a second antibody against hCG which is labeled for detection.

21. The method of claim 20 wherein said second antibody is labeled with an enzyme.

22. The method of claim 20 wherein said second antibody is attached to a polymeric particle having a detectable tracer material.

23. The method of claim 19 wherein said immunoreactive species is a monoclonal antibody.

24. The method of claim 19 wherein said polymer particle has a tracer associated therewith.

25. The method of claim 24 wherein said tracer is totally within said particle.

26. The method of claim 17 wherein said polymer is represented by the formula:
wherein -A- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers, -B- represents recurring units derived from one or more ethylenically unsaturated monomers having a reactive group selected from the group consisting of activated 2-substituted ethylsulfonyl, vinylsul-fonyl or an active halogen atom, -D- represents recurring units derived from one or more ethylenically unsaturated monomers other than those represented by -A- or -B-, x is from 0 to about 99.9 mole percent, y is from about 0.1 to about 100 mole percent, and z is from 0 to about 20 mole percent.

27. The method of claim 17 for the determination of a human retrovirus.

28. A method for the determination of a compound of biological interest in an aqueous liquid comprising:
A. contacting said liquid with a water-insoluble reagent comprising:
a core/shell polymeric particle in which the shell comprises a polymer which is represented by the formula:
wherein -A- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers, -B- represents recurring units derived from one or more ethylenically unsaturated monomers having a reactive group selected from the group consisting of activated 2-substituted ethylsulfonyl, vinylsul-fonyl or an active halogen atom, -D- represents recurring units derived from one or more ethylenically unsaturated monomers other than those represented by -A- or -B-, x is from 0 to about 99.9 mole percent, y is from about 0.1 to about 100 mole percent, and z is from 0 to about 20 mole percent, said particle being covalently attached through said reactive groups on the outer surface of said shell to avidin or a derivative thereof, B. forming a reaction product of said reagent with a biotinylated reagent comprised of biotin or a derivative thereof attached to a first receptor molecule for said biological compound so as to form an insoluble specific binding complex between avidin and said biotinylated reagent, C. prior to, simultaneously with or sub-sequent to said contacting step A, contacting said biological compound with a second receptor molecule which is capable of participating in a specific binding reaction with said biological compound but which is not reactive with said first receptor molecule, said second receptor being labeled with a detectable tracer material, so as to form a labeled insoluble complex, and D. detecting said labeled insoluble complex.

29. The method of claim 28 wherein said biological compound is an antigen and said first and second receptors are distinct antibodies to said antigen.

30. The method of claim 28 wherein said biological compound is an antibody, said first receptor is an antigen reactive with said compound, and said second receptor is an antibody directed against said compound.